Secreted Frizzled-Related Proteins and Wnt Signaling

我的实验室的重点已经扩大到包括分泌的卷曲相关蛋白（sFRPs）和细胞对Wnt刺激的反应的研究。我们已经开始纯化Wnt蛋白，不仅提供试剂以促进sFRP功能的分析，而且促进Wnt活性和相关信号传导的研究。确定Wnt/Frizzled（Fz）/sFRP相互作用的特异性是我们的主要目标之一。目前，我们已经纯化了四种重组sFRPs，并在分离三种Wnt蛋白方面取得了进展。我们还鉴定了具有低背景Wnt/Fz表达的细胞系，并将三种Fz分别引入这些细胞中以帮助我们研究Wnt/Fz/sFRP结合和信号传导。最近，我们发起了一项合作，以探讨Cripto作为一种新型Wnt辅助受体在经典/？连环蛋白途径我们开发了一个方便的模型系统来检查Wnt依赖的细胞运动性和相关的细胞形状变化。我们证明，RhoA激活是重要的细胞迁移，而？连环蛋白信号传导不是，并且Dishevelled-2（Dvl-2）在transwell测定中作为Wnt活性的介体具有主要作用。这导致了对Dvl调节方面的强调，特别是磷酸化，其被认为对于非经典信号传导和细胞运动性是重要的。我们还观察到Wnt-3a刺激纤连蛋白（FN）原纤维的组装，这是一种可能有助于细胞扩散和运动的现象。我们对sFRP-1的长期研究在各种背景下继续进行。我们已经确定SFRP 1启动子中特定序列的甲基化破坏了核因子的结合，这可能有助于在几种癌症中记录的基因的表观遗传沉默。已经开发了一种Sfrp 1 null小鼠模型，我们正在与多个实验室分享它，以更好地了解sFRP-1的生理活性。在各种实验系统中，我们一直在比较和对比sFRP-1和Dickkopf-1（Dkk-1）的效果。当两者都抑制？-我们的研究结果表明，Dkk-1可能会增强非经典信号的情况下，它是由sFRP-1阻断。除了sFRP-1作为Wnt信号传导调节剂的公认作用外，我们还探索了它具有新的结合伴侣和其他作用机制的可能性。使用噬菌体展示肽库，我们确定了sFRP-1的结合基序，这是不存在于Wnt蛋白，并可能提供线索，其他蛋白质-蛋白质相互作用。我们一度认为，这种相互作用可能是sFRP-1抑制破骨细胞形成的基础，我们与Matthew吉莱斯皮博士合作证明了这种作用。RANKL是破骨细胞生成的关键介质，含有类似的基序，并在ELISA实验中与sFRP-1结合。然而，随后的工作表明sFRP-1抑制活性不需要与RANKL直接相互作用。总之，我们的sFRP和Wnt研究将有助于更好地了解它们在癌症，发育和组织稳态中的作用。

The focus of my lab has broadened to include both the study of secreted Frizzled-related proteins (sFRPs) and cellular responses to Wnt stimulation. We have embarked on the purification of Wnt proteins, not only to provide reagents to facilitate the analysis of sFRP function, but to foster investigation of Wnt activity and associated signaling. Defining the specificity of Wnt/Frizzled(Fz)/sFRP interactions is one of our primary goals. Currently, we have purified four recombinant sFRPs and have made progress in the isolation of three Wnt proteins. We also identified a cell line with a low background of Wnt/Fz expression, and separately introduced three Fzs into these cells to assist our study of Wnt/Fz/sFRP binding and signaling. Recently, we initiated a collaboration to explore the alleged role of Cripto as a novel Wnt co-receptor functioning in the canonical/?-catenin pathway. We developed a convenient model system to examine Wnt-dependent cell motility and associated changes in cell shape. We demonstrated that RhoA activation is important for cell migration, while ?-catenin signaling is not, and Dishevelled-2 (Dvl-2) had a predominant role as a mediator of Wnt activity in a transwell assay. This has led to an emphasis on aspects of Dvl regulation, particularly phosphorylation, thought to be important for non-canonical signaling and cell motility. We also have observed that Wnt-3a stimulates the assembly of fibronectin (FN) fibrils, a phenomenon that likely contributes to cell spreading and movement. Our longstanding study of sFRP-1 has continued in a variety of contexts. We have determined that methylation of specific sequences in the SFRP1 promoter disrupts binding of nuclear factors, which may contribute to epigenetic silencing of the gene that has been documented in several cancers. An Sfrp1 null mouse model has been developed, and we are sharing it with multiple labs to better understand the physiological activities of sFRP-1. In various experimental systems, we have been comparing and contrasting the effects of sFRP-1 and Dickkopf-1 (Dkk-1). While both inhibit the ?-catenin pathway, our results suggest that Dkk-1 might enhance non-canonical signaling in situations where it is blocked by sFRP-1. Besides the well-established role of sFRP-1 as a regulator of Wnt signaling, we have explored the possibility that it has novel binding partners and additional mechanisms of action. Using a phage-display peptide library, we identified a binding motif for sFRP-1 that is not present in Wnt proteins and might provide a clue to other protein-protein interactions. At one point, we thought that such interactions could be the basis for sFRP-1 inhibition of osteoclast formation, an effect which we demonstrated in collaboration with Dr. Matthew Gillespie. RANKL, a key mediator of osteoclastogenesis, contains a similar motif and bound to sFRP-1 in ELISA experiments. However, subsequent work suggests that sFRP-1 inhibitory activity does not require a direct interaction with RANKL. Taken together, our sFRP and Wnt studies will contribute to a better understanding of their roles in cancer, development and tissue homeostasis.